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At least 217 records · Page 12Linked to original sources
PubMed1995

Quality assurance program for cyclosporin G (OG37-325).

Cyclosporine-G (CsG) (OG37-325) an analogue of cyclosporine-A (CsA) is presently undergoing clinical trials. As therapeutic monitoring of CsG was an integral part of these evaluations, the performance of laboratories measuring the drug was assessed through an external quality assurance program, which included all North American centers participating in the trials. The assays used by participating centers were high-performance liquid chromatography (HPLC) (n = 3), radioimmunoassay (RIA) (n = 5), and fluorescence polarization immunoassay (FPIA) (n = 24). The latter two assays, developed for measurement of CsA, were adapted for measurement of CsG. The FPIA was the most precise, with the largest proportion of laboratories reporting coefficients of variation (CNs) of < 10%. RIA was the least accurate method, with approximately 74% of results differing by > 20% from target values. FPIA and RIA methods exhibited mean recoveries of CsG of 112 and 129%, respectively. The ranking of the specificity of the assays from measurement of parent drug was HPLC > RIA > FPIA. FPIA and RIA produced values that were, on average, approximately 60% higher than those reported by HPLC in pooled whole blood specimens obtained from renal transplant patients. It is recommended that if CsG is approved for routine clinical use, then assays developed specifically for measurement of the drug be used.

Bias↗
PubMed1999

Led-NPF-1 stimulates ovarian development in locusts.

For more than a decade, immunohistochemical results on FMRFamide related peptides (FaRP's) have been reported extensively, suggesting many possible roles for these peptides associated with behavioural and physiological events as well as reproduction. This study provides a clear effect in vivo of members of this family of insect neuropeptides. The effect of two neuropeptide F-related peptides from the Colorado potato beetle, Leptinotarsa decemlineata, Led-NPF-1 and Led-NPF-2 as well as the locusts myotropins, Lom-PK-1, Lom-PK-2 and Lom-SK, was screened in an ovarian development assay in the African migratory locust and the grey fleshfly, Neobellieria bullata. Led-NPF-1 (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH2) was shown to be a potent gonadostimulin in Locusta migratoria, but not in Neobellieria bullata. A minimal dose of 0.05 microg of Led-NPF-1 per animal, every 12 h, during 5 consecutive injections into 6 day old virgin females, could accelerate egg development. Higher doses of prolonged injections were demonstrated to be even more potent in the ovarian development assay. Led-NPF-2 (Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH2) was far less active. The other tested peptides scored no reproducible effect what so ever on ovarian growth, in locusts, nor in flies. The gonadotropic action of a NPF-like peptide on oocyte growth implies a complex regulation of oogenesis in the locust and adds to our knowledge of insect neuroendocrinology in general. The results also suggest that a peptide of similar sequence also resides in the locust.

Animals↗
PubMed2003

Recombinant baculoviruses used to study estrogen receptor function in human osteosarcoma cells.

We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.

Animals↗
PubMed2003

Comparison of glycoprotein B (gB) variants of the elephant endotheliotropic herpesvirus (EEHV) isolated from Asian elephants (Elephas maximus).

The recently described elephant endotheliotropic herpesviruses (EEHV) have been associated with the deaths of numerous captive elephants. A proposed tool for the detection of EEHV infection in elephants is the PCR-based screening for EEHV-DNA in whole blood samples. Unfortunately, this detection method has only been successful in post-mortem analyses or in animals already displaying clinical signs of EEHV disease, thus rendering this method unsuitable for identification of carrier elephants. Here, we focus on glycoprotein B (gB) for serologic assay development, since gB is an envelope protein known to induce a neutralising antibody response in other herpesvirus infections. We sequenced the entire gB gene from five Asian elephants with EEHV, representing four different gB variants. Computer-aided methods were used to predict functionally important regions within EEHVgB. An extra-cytoplasmic region of 153 amino acids was predicted to be under positive selection and may potentially contain antigenic determinants that will be useful for future serologic assay development.

Amino Acid Sequence↗
PubMed2005

An enrichment broth culture-duplex PCR combination assay for the rapid detection of enterotoxigenic Clostridium perfringens in fecal specimens.

An enrichment broth culture-duplex PCR combination assay was devised to identify Clostridium perfringens directly from fecal samples. The method consists of a combination of short enrichment of samples in selective media, DNA isolation, and performing duplex PCR using two pairs of primers which identify C. perfringens strains that harbor the virulence enterotoxin gene. Comparison of two selective enrichment media and two incubation temperatures showed that the reinforced clostridial medium with neomycin was better than the fluid thioglycollate medium with neomycin (p<0.001); and incubation at 37 degrees C vs 45 degrees C showed no statistically significant difference (p=0.238). The optimal short time for pre-enrichment culture was 4 hours. The developed assay was applied to detect phospholipase C (plc) and enterotoxin (cpe) genes for C. perfringens in feces inoculated artificially with enterotoxigenic C. perfringens. The method could detect both gene products in samples inoculated with a minimum of 10(4) CFU per ml. When the method was applied to detect enterotoxigenic C. perfringens in 198 diarrhea patients, C. perfringens was found in 121 samples; 7 out of 121 samples were positive for both plc and cpe (prevalence of 5.8%). These results indicate that the developed assay was a suitable method for the rapid and specific detection of enterotoxigenic C. perfringens directly in fecal specimens of diarrhea patients, which will assist epidemiological investigations of food poisoning outbreaks and quality control of food products.

Base Sequence↗
PubMed2026

Disruption of Polycystin Ciliary Localization and Channel Function by Autosomal Dominant Polycystic Kidney Disease-Causing Polycystin-1 Variants.

KEY POINTS: We developed assays to measure genetic variant effects on polycystin-1, the protein mutated in most autosomal dominant polycystic kidney disease. All tested pathogenic variants disrupted either polycystin-1 ciliary trafficking or channel function. Trafficking and channel function of some pathogenic variants was restored by low temperature culture to promote polycystin folding. BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of ADPKD, limited mechanistic understanding has hindered therapeutic development. Most ADPKD is caused by loss-of-function variants in polycystin-1 (PC1). METHODS: We developed assays that quantify the effect of nontruncating variants on PC1 ciliary localization, membrane trafficking, and polycystin channel function. RESULTS: We evaluated 29 nontruncating variants in PC1 and found that pathogenic variants disrupt two molecular phenotypes: ( 1 ) localization of PC1 at the primary cilium or ( 2 ) polycystin ion channel activity. Ciliary localization of a subset of polycystin variants was restored when cells were cultured at low temperature. A subset of variants with localization restored by low temperature formed functional channels. CONCLUSIONS: This study demonstrated that disruptions in polycystin ciliary trafficking and channel function are common causes of ADPKD. Defects in ciliary trafficking and channel function can be rescued for a subset of pathogenic variants, establishing a foundation for polycystin-targeted therapies in ADPKD.

Polycystic Kidney, Autosomal Dominant↗
PubMed1974

Improvements of the nitrite color development in assays of nitrate reductase by phenazine methosulfate and zinc acetate.

Nitrate reductase activity is most commonly assayed by measurement of product formation. Excess NADH and factor(s) present in the enzyme extract that interfere with the diazotization and azo color complex of nitrite cause a depression of apparent nitrate reductase activity. Two postassay treatments were found that markedly enhanced the extent of nitrite color formation and apparent nitrate reductase activity. The procedure involves stopping the reaction with zinc acetate (50 mumoles per ml of reaction mix), followed by removal of the precipitate by centrifugation. Presumably the zinc acetate removes extract factor(s) that interfere with color development, because it does not remove the NADH. Phenazine methosulfate (15 nmoles per ml of reaction mix) is added to aliquots of the supernatant and allowed to stand for 20 min at 30 C to oxidize the residual NADH before color development.

Journal Article↗
PubMed2005

Development of ELISA and immunochromatographic assay for the detection of neomycin.

BACKGROUND: Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields. METHODS: Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk. RESULTS: No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50-200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. CONCLUSIONS: Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA.

Animals↗
PubMedDate not supplied

Collagen cross-links. Synthesis of immunoreagents for development of assays for deoxypyridinoline, a marker for diagnosis of osteoporosis.

(+)-Deoxypyridinoline (Dpd, 2) is a cross-link of bone collagen, which is released and excreted in urine during process of bone resorption. It has been shown that this bone collagen degradation product, Dpd (2) is a useful marker for diagnosis of osteoporosis and other metabolic bone diseases. In this paper, the design and synthesis of two immunogens (3, 4) via of conjugation of succinimidyl ester (14) to carrier protein, bovine serum albumin, or keyhole limphet hemocyanin, was presented. Additionally, fluorescent (5) and chemiluminescent (6) tracers were prepared from (-)-acid (13) via in situ activation and subsequent reaction with 6-Fln-CH(2)NH(2) (17) or Acr-NH(2) (18) and hydrolysis. The key hapten (-)-acid (13) was prepared by quaternization of 3-hydroxypyridine derivative (S,S)-(-)-11 with iodide (S)-(-)-10 followed by selective hydrolysis. These immunreagents (immunogens 3 and 4 and tracers 5 and 6) are useful for the development of sensitive and high throughput immunoassays, such as FPIA and CLIA for Dpd (2).

Adjuvants, Immunologic↗
PubMed2005

Quantitation of feline leukaemia virus viral and proviral loads by TaqMan real-time polymerase chain reaction.

Feline leukaemia virus (FeLV) infection in cats is not only of veterinary importance but also a well-acknowledged animal model for studying the pathogenesis of retroviral disease. After virus exposure, different courses and outcomes of FeLV infection may prevail; they have been associated with cellular and humoral immune responses and the FeLV proviral load in peripheral blood. We hypothesized that the plasma viral RNA load might be an additional relevant indicator for the infection outcome. To quantify these loads, a real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed. The assay amplifies FeLV-A, -B, and -C as some naturally infected cats could not be identified with a FeLV-A-based assay previously. The assay was applied to determine plasma FeLV RNA loads in cats infected both naturally and experimentally with FeLV. In addition, an improved real-time PCR assay for quantitation of FeLV proviral loads is described. The assays developed were more sensitive than ELISA and virus isolation in the early phase of infection. In addition, PCR allows the identification of provirus carriers that have overcome antigenaemia. Thus, for most effective detection of FeLV exposure and characterization of the infection in a cat, PCR assays are recommended as diagnostic tools.

Animals↗
PubMed2005

Development of ELISA and immunochromatographic assay for the detection of gentamicin.

Competitive direct enzyme-linked immunosorbent assay (ELISA) and the immunochromatographic assay were developed using a monoclonal antibody to detect gentamicin in the animal plasma and milk. No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA, indicating that the antibody is highly specific for gentamicin. On the basis of the standard curves, the detection limits were determined to be 0.9 ng/mL in phosphate-buffered saline (PBS), 1.0 ng/mL in plasma, and 0.5 ng/mL in milk, respectively. Recoveries of gentamicin from spiked plasma and milk at levels of 25-100 ng/mL ranged from 85 to 112%. The concentration of intramuscularly injected gentamicin was successfully monitored in the rabbit plasma through competitive direct ELISA. The detection limits were estimated to be about 6 ng/mL of gentamicin in PBS, plasma, and milk using the colloidal gold-based immunochromatographic assay, which is suitable for the simple screening of gentamicin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could complement each other as well as veterinary field and laboratory findings.

Animals↗
PubMedDate not supplied

Evaluation of lipid peroxidation in red blood cells by monitoring the uptake of sucrose and phenol red.

Red blood cells (RBCs) are prone to lipid peroxidation by virtue of their function as oxygen carriers, and also because of their lipid composition. Malondialdehyde (MDA) content using thiobarbituric reagent is widely used to quantify lipid peroxidation. In this study we compare MDA assay with a newly developed assay that evaluates the uptake of sucrose and phenol red into RBCs under peroxidative stress. Both sucrose and phenol red uptake show significantly higher correlation with incubation time compared with MDA assay. Furthermore, phenol red uptake into RBCs on treatment with H(2)O(2) has a direct linear proportional relationship, whereas it is hyperbolic with MDA. The assay also clearly shows that uptake of sucrose or phenol red is specific for intact cells (RBCs) prior to hemolysis. Assay validation is carried out by using known lipid peroxidation-causing agents, such as ferrous ions, and also by using peroxidation inhibitors such as alpha-tocopherol. This new method can be applied efficiently to evaluate lipid peroxidation in RBCs as well as other cells and tissues.

Absorption↗
PubMed1992

Assessment of former and newly developed HBV assays in a Third World setting.

Newly available HBV serological assays have not been established routinely in most underdeveloped countries. Utilizing enzyme-immune assays to determine the presence of pre-S1 antigen and anti-pre-S2, and using two conventional hybridization techniques and the PCR assay to detect HBV-DNA, we studied 30 HBsAg chronic carriers and as a reference group 10 subjects whose only HBV routine marker was anti-HBc. Seventy-nine percent of the HBeAg positive carriers showed detectable HBV-DNA by a non-radioactive slot-blotting technique. The PCR assay was more sensitive than the slot-blotting technique, detecting HBV-DNA in anti-HBe positive patients with moderate or normal ALT activity. Pre-S1 antigen was mostly related to the presence of HBsAg and anti-pre-S2 was associated with active viremic state, increased ALT activity (ranges 51 to 640 IU/L), and with self-limited HBV infection. The presence of HBV-DNA in the group with anti-HBc only was detectable solely by the PCR assay. For an underdeveloped country the addition of a PCR assay or pre-S/anti-pre-S protein tests to the current assessment procedures of HBV chronic infection should be used only in selective cases. HBeAg/anti-HBe serological evaluation and HBV-DNA detection by a non-isotopic conventional hybridization technique still remain as useful tools to screen initially for the presence of viremia in chronic HBsAg carriers. The presence of HBV-DNA in individuals with anti-HBc only suggests that anti-HBc screening should be maintained and expanded to all the blood banks of less industrialized countries where the rate of HBV infection in apparently healthy people tends to be high.

Adult↗
PubMed1995

Characterization of a specific monoclonal antibody 9F5-3a and the development of assay system for oxidized HDL.

We obtained a monoclonal antibody 9F5-3a against oxidative low-density lipoprotein (LDL) modified with CuSO4 and established a sandwich ELISA for detection of oxidized high-density lipoprotein (oxHDL). The 9F5-3a was reacted strongly with oxHDL and to a lesser degree with oxLDL and LDL. In contrast, little or no reactivity was found with HDL. When the generation of oxHDL was limited by the addition of alpha-tocopherol and catalase, reactivity to 9F5-3a was reduced. Incubation of oxHDL with excess lyso-phosphatidylcholine (lyso-PC) also reduced immunoreactivity, but not by only lyso-PC. These results suggested that the epitope is possibly associated with oxHDL-linked lyso-PC induced mainly by the hydroxyl radical.

Animals↗
PubMed1986

Radioimmunoassays for fish tail neuropeptides: I. Development of assay and measurement of immunoreactive urotensin I in Catostomus commersoni brain, pituitary, and plasma.

Antisera were raised in rabbits using highly purified urotensin I (UI) or UI (4-41) conjugated with high-molecular-weight proteins. Antiserum 2U4, at a final dilution of 1:300,000, gave 50% binding to iodinated UI (specific activity 40-120 microCi/micrograms). Carp (Cyprinus carpio) and sucker (Catostomus commersoni) UI peptides cross-reacted completely with the antiserum. Results from cross-reactivity tests using various tryptic and Staphylococcus aureus protease fragments of UI indicated that the antigenic sites of the main antibody(ies) are directed against a part sequence in the C-terminal region of the peptide although antibodies with lower concentration or affinity, recognizing N-terminal region(s) of the peptide, are also present. No cross-reactivity was seen with insulin, secretin, vasoactive intestinal peptide or a structural UI-homolog, the ovine hypothalamic corticotropin-releasing factor, up to molar concentrations. Sauvagine, another structurally homologous peptide from frog skin, showed only 0.1% cross-reactivity. The urophysis-specific proteins, urophysins B and D, showed a low degree of cross-reactivity. Assays of serial dilutions of urophysis extracts from teleostean species yielded parallel displacement curves, except in the case of the pacific goby, Gillichthys mirabilis. Varying concentrations of immunoreactive UI were present in different regions of C. commersoni brain, spinal cord, and pituitary, and also in plasma. The measurement of 17.5 +/- 2.1 fmol UI/ml of plasma indicates that this assay may be used for the study of circulating UI peptide(s).

Amino Acid Sequence↗